We propose to continue to utilize molecular genetic techniques to analyze the structure, function, and control of expression of the Na/K ATPase. During the previous granting period we have developed the capacity to assay the function of the mouse Na/K ATPase alpha subunit gene by the ability to confer ouabain resistance to monkey CV-1 cells. We now propose to extend these studies by addressing the following specific aims. We will: 1. Analyze the interaction between the enzyme and cardiac glycosides using molecular genetic techniques including site specific mutagenesis and formation of chimeras between ouabain sensitive and ouabain resistant forms of the enzyme. 2. Develop oligopeptide antibodies against specific sites on the alpha and beta subunits. 3. Use in vitro translation to produce alpha and beta subunits separately and attempt to reconstitute enzyme activity. 4. Use site specific mutagenesis to identify specific functional sites on the alpha subunit. 5. Determine the interaction between levels of Na/K ATPase expression and cell viability. 6. Develop an assay for the in vivo expression of the beta subunit of the enzyme. 7. Develop a system for studying subcellular localization of the enzyme.